Mitochondrial Dosimeter

ABSTRACT

Mitochondrial mutations occur as a product of contact of a person with an environmental pollutant. Mitochondrial mutations are readily detectable in body fluids. Measurement of mitochondrial mutations in body fluids can be used as a dosimeter to monitor exposure to the environmental pollutant. Mitochondrial mutations can also be detected in cancer patients. Probes and primers containing mutant mitochondrial sequences can be used to monitor patient condition.

The U.S. Government retains certain rights in this invention due tofunding as provided by grant CA43460 awarded by the National Institutesof Health.

This application is a continuation of Ser. No. 10/601,692 filed Jun. 23,2003, is a division of Ser. No. 09/525,906 filed Mar. 15, 2000, is acontinuation-in-part of application Ser. No. 09/377,856 filed Aug. 20,1999, which claims priority to provisional application Ser. No.60/097,307 filed Aug. 20, 1998. The disclosure of these priorapplications is expressly incorporated herein.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the field of environmental toxicology, inparticular to methods for measuring the effects of environmental toxins.

BACKGROUND OF THE INVENTION

The human mitochondrial (mt) genome is small (16.5 kb) and encodes 13respiratory chain subunits, 22 tRNAs and two rRNAs. Mitochondrial DNA ispresent at extremely high levels (10³-10⁴ copies per cell) and the vastmajority of these copies are identical (homoplasmic) at birth (1).Expression of the entire complement of mt genes is required to maintainproper function of the organelle, suggesting that even slightalterations in DNA sequences could have profound effects (2). It isgenerally accepted that mtDNA mutations are generated endogenouslyduring oxidative phosphorylation via pathways involving reactive oxygenspecies (ROS), but they can also be generated by external carcinogens orenvironmental toxins. These mutations may accumulate partially becausemitochondria lack protective histones and highly efficient DNA repairmechanisms as seen in the nucleus (3). Recently several mtDNA mutationswere found specifically in human colorectal cancer (4).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods ofmonitoring exposure of a person to an environmental pollutant.

It is another object of the present invention to provide a kit formonitoring exposure of a person to environmental pollutants.

It is an object of the invention to provide methods to aid in thedetection of cancer or metastasis.

It is an object of the invention to provide probes and primers fordetecting mitochondrial mutations.

It is an object of the invention to provide a method to aid in detectingthe presence of tumor cells in a patient.

These and other objects of the invention are achieved by providing oneor more of the embodiments described below. In one embodiment a methodis provided for monitoring exposure of a person to an environmentalpollutant. The presence of one or more mutations in mitochondrial DNA(mtDNA) in a body fluid of a person exposed to an environmentalpollutant is determined at two or more time points. The amounts ofmutations in mtDNA at different time points are compared. The amount ofmutations correlates with amount of exposure to the environmentalpollutant.

According to another embodiment another method is provided formonitoring exposure of a person to an environmental pollutant. Theprevalence of one or more mutations in mitochondrial DNA (mtDNA) in abody fluid of a person exposed to an environmental pollutant ismeasured. A measured prevalence of one or more mutations in mtDNA ofgreater than 1% indicates clonal expansion of cells which harbor the oneor more mutations in the person.

According to still another embodiment of the invention a method isprovided for monitoring exposure of a person to an environmentalpollutant. One or more mutations in a D-loop of mitochondrial DNA(mtDNA) in a body fluid of a person exposed to an environmentalpollutant are measured. The number of mutations in mtDNA correlates withexposure to the environmental pollutant.

According to yet another embodiment of the invention a kit is provided.The kit comprises one or more primers which hybridize to a mitochondrialD-loop for making a primer extension product. In addition, the kitcontains written material identifying mutations which are found in theD-loop as a result of exposure to one or more environmental pollutants.

According to another embodiment of the invention an oligonucleotideprobe is provided. The probe comprises a sequence of at least 10contiguous nucleotides of a human mitochondrial genome. The probe canoptionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 suchcontiguous nucleotides. The oligonucleotide comprises a mutationselected from the group consisting of: a mutation selected from thegroup consisting of: T→C at nucleotide 114; ΔC at nucleotide 302; C→A atnucleotide 386; insert T at nucleotide 16189; A→C at nucleotide 16265;A→T at nucleotide 16532; C→T at nucleotide 150; T→C at nucleotide 195;ΔC at nucleotide 302; C→A at nucleotide 16183; C→T at nucleotide 16187;T→C at nucleotide 16519; G→A at nucleotide 16380; G→A at nucleotide 75;insert C at nucleotide 302; insert CG at nucleotide 514; T→C atnucleotide 16172; C→T at nucleotide 16292; A→G at nucleotide 16300; A→Gat nucleotide 10792; C→T at nucleotide 10793; C→T at nucleotide 10822;A→G at nucleotide 10978; A→G at nucleotide 11065; G→A at nucleotide11518; C→T at nucleotide 12049; T→C at nucleotide 10966; G→A atnucleotide 11150; G→A at nucleotide 2056; T→C at nucleotide 2445; T→C atnucleotide 2664; T→C at nucleotide 10071; T→C at nucleotide 10321; T→Cat nucleotide 12519; Δ 7 amino acids at nucleotide 15642; G→A atnucleotide 5521; G→A at nucleotide 12345; T→C substitution at position710; T→C substitution at position 1738; T→C substitution at position3308; G→A substitution at position 8009; G→A substitution at position14985; T→C substitution at position 15572; G→A substitution at position9949; T→C substitution at position 10563; G→A substitution at position6264; A insertion at position 12418; T→C substitution at position 1967;T→A substitution at position 2299; and G→A at nucleotide 3054.

According to another aspect of the invention an oligonucleotide primeris provided. It comprises a sequence of at least 10 contiguousnucleotides of a human mitochondrial genome. The primer can optionallycontain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguousnucleotides. The oligonucleotide comprises a mutation selected from thegroup consisting of: a mutation selected from the group consisting of:T→C at nucleotide 114; ΔC at nucleotide 302; C→A at nucleotide 386;insert T at nucleotide 16189; A→C at nucleotide 16265; A→T at nucleotide16532; C→T at nucleotide 150; T→C at nucleotide 195; ΔC at nucleotide302; C→A at nucleotide 16183; C→T at nucleotide 16187; T→C at nucleotide16519; G→A at nucleotide 16380; G→A at nucleotide 75; insert C atnucleotide 302; insert CG at nucleotide 514; T→C at nucleotide 16172;C→T at nucleotide 16292; A→G at nucleotide 16300; A→G at nucleotide10792; C→T at nucleotide 10793; C→T at nucleotide 10822; A→G atnucleotide 10978; A→G at nucleotide 11065; G→A at nucleotide 11518; C→Tat nucleotide 12049; T→C at nucleotide 10966; G→A at nucleotide 11150;G→A at nucleotide 2056; T→C at nucleotide 2445; T→C at nucleotide 2664;T→C at nucleotide 10071; T→C at nucleotide 10321; T→C at nucleotide12519; Δ 7 amino acids at nucleotide 15642; G→A at nucleotide 5521; G→Aat nucleotide 12345; T→C substitution at position 710; T→C substitutionat position 1738; T→C substitution at position 3308; G→A substitution atposition 8009; G→A substitution at position 14985; T→C substitution atposition 15572; G→A substitution at position 9949; T→C substitution atposition 10563; G→A substitution at position 6264; A insertion atposition 12418; T→C substitution at position 1967; T→A substitution atposition 2299; and G→A at nucleotide 3054.

Another aspect of the invention is a method to aid in detecting thepresence of tumor cells in a patient. The presence of a single basepairmutation is detected in a mitochondrial genome of a cell sample of apatient. The mutation is found in a tumor of the patient but not innormal tissue of the patient. The tumor is not a colorectal tumor. Thepatient is identified as having a tumor if one or more single basepairmutations are determined in the mitochondrial genome of the cell sampleof the patient.

Yet another embodiment of the invention is provided by another method toaid in detecting the presence of tumor cells in a patient. The presenceof a mutation is determined in a D-loop of a mitochondrial genome of acell sample of a patient. The mutation is found in a tumor of thepatient but not in normal tissue of the patient. The patient isidentified as having a tumor if one or more single basepair mutationsare determined in the mitochondrial genome of the cell sample of thepatient.

According to still another aspect of the invention a method is providedto aid in detecting the presence of tumor cells in a patient. Thepresence of a single basepair mutation is determined in a mitochondrialgenome of a cell sample of a patient. The mutation is found in a cancerof the patient but not in normal tissue of the patient. The cancer isselected from the group of cancers consisting of: lung, head and neck,bladder, brain, breast, lymphoma, leukaemia, skin, prostate, stomach,pancreas, liver, ovarian, uterine, testicular, and bone. The patient isidentified as having a tumor if one or more single basepair mutationsare determined in the mitochondrial genome of the cell sample of thepatient.

According to still another aspect of the invention a method is providedto aid in detecting the presence of tumor cells in a patient. Thepresence of a single basepair mutation is determined in a mitochondrialgenome of a cell sample of a patient. The mutation is found in a tumorof the patient but not in normal tissue of the patient. The cancer isselected from the group of cancers consisting of: lung, head and neck,and bladder. The patient is identified as having a tumor if one or moresingle basepair mutations are determined in the mitochondrial genome ofthe cell sample of the patient.

Another embodiment of the invention provides a method to aid indetecting the presence of tumor cells in a patient. The presence of amutation in a mitochondrial genome of a cell sample of a patient isdetermined. The mutation is selected from the group consisting of: T→Cat nucleotide 114; ΔC at nucleotide 302; C→A at nucleotide 386; insert Tat nucleotide 16189; A→C at nucleotide 16265; A→T at nucleotide 16532;C→T at nucleotide 150; T→C at nucleotide 195; ΔC at nucleotide 302; C→Aat nucleotide 16183; C→T at nucleotide 16187; T→C at nucleotide 16519;G→A at nucleotide 16380; G→A at nucleotide 75; insert C at nucleotide302; insert CG at nucleotide 514; T→C at nucleotide 16172; C→T atnucleotide 16292; A→G at nucleotide 16300; A→G at nucleotide 10792; C→Tat nucleotide 10793; C→T at nucleotide 10822; A→G at nucleotide 10978;A→G at nucleotide 11065; G→A at nucleotide 11518; C→T at nucleotide12049; T→C at nucleotide 10966; G→A at nucleotide 11150; G→A atnucleotide 2056; T→C at nucleotide 2445; T→C at nucleotide 2664; T→C atnucleotide 10071; T→C at nucleotide 10321; T→C at nucleotide 12519; Δ 7amino acids at nucleotide 15642; G→A at nucleotide 5521; G→A atnucleotide 12345; T→C substitution at position 710; T→C substitution atposition 1738; T→C substitution at position 3308; G→A substitution atposition 8009; G→A substitution at position 14985; T→C substitution atposition 15572; G→A substitution at position 9949; T→C substitution atposition 10563; G→A substitution at position 6264; A insertion atposition 12418; T→C substitution at position 1967; T→A substitution atposition 2299; and G→A at nucleotide 3054. The patient is identified ashaving a tumor if one or more mutations are determined in themitochondrial genome of the cell sample of the patient.

These and other embodiments provide the art with non-invasive tools formonitoring exposure to and the effects of environmental pollutants onthe human body as well as early detection methods for cancer andmetastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of a linearized mt genome. Hatched barsindicate the regions sequenced in this study and solid bars indicate thepositions of tRNAs (transfer RNAs). rRNA=ribosomal RNA, ND=NADHdehydrogenase, COX=cytochrome c oxidase, Cyt b=cytochrome b, ATPase=ATPsynthase.

FIG. 2. Sequence detection of mutated mtDNAs in samples from tumors andbodily fluids. (FIG. 2A) The mt mutation was analyzed by directsequencing of the tumor (T), normal (N), and corresponding urine (U)DNAs of bladder cancer patient #799. The arrow indicates a singlenucleotide change (G(A) at 2056 np in the 16S rRNA gene. (FIG. 2B andFIG. 2C) Examples of somatic mutations in head and neck cancers. Bothmutations at 16172 np (B) and 10822 np (C) were detected from saliva (S)samples from patients #1680 and #1708, respectively. (FIG. 2D) MutatedmtDNA at 2664 np was not detected by sequence analysis in the paired BALfluid (B), obtained from lung cancer patient #898.

FIG. 3. Oligonucleotide-mismatch ligation assay (22) to detect mtDNAmutations in BAL. The arrows identify mutated mt sequences at 12345 npwithin tRNA (FIG. 3A) and at 2664 np (FIG. 3B) within 16S rRNA in thetumor DNA. More dilute signals are seen in the corresponding BAL (B)samples with no detectable signal from the paired normal (N) tissue.

FIG. 4. Highly enriched mutated mtDNA in BAL samples from lung cancerpatients. Oligonucleotide (oligo) specific hybridization detected ˜2000plaques containing WT p53 clones in the BAL from patient #1113, and onlytwo plaques (2/2000=0.1%) with the p53 gene mutation (FIG. 4A) werefound in the primary tumor. The same BAL sample demonstrated a muchgreater enrichment of mutated mtDNA; 445 plaques contained mtDNAmutations (FIG. 4A) at 16159 np (445/2000=22.3%; 220-fold) compared toapproximately 1500 WT clones. A similar enrichment was seen in patient#1140 where oligo specific hybridization detected 12 p53 mutant plaquesamong 437 WT clones (2.7%, FIG. 4B), while mutant mtDNA at 16380 np(FIG. 4B) represented over 50% of the plaques (52.3%, 460/880; 19-fold)amplified from mtDNA.

FIG. 5. Pseudoclonal selection of mtDNA. A mitochondrial genome gainssome replicative advantage due to a somatic mutation (such as in theD-loop region), leading to a dominant mitochondrial genotype (step 1).This mitochondrion can gain additional replicative advantage throughnuclear influences: for example, a mutated sequence gains a higherbinding affinity to nuclear-encoded mitochondrial trans-acting factors(step 2). Due to its stochastic segregation together with the clonalexpansion of a neoplastic cell driven by nuclear mutations, mutatedmitochondria overtake the entire population of tumor cells (step 3).

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides a summary of mutations in mitochondria of colorectaltumors.

Table 2 provides a summary of mutations in mitochondria of bladder,lung, head and neck tumors.

Table 3 provides a summary of new polymorphisms in mitochondria ofbladder, lung, head and neck tumors.

DETAILED DESCRIPTION

It is a discovery of the present inventors that mitochondrial DNAmutations can be monitored non-invasively and sensitively and used as anindicator of environmental pollutants. It is shown below that thesemutations are more prevalent in body samples than nuclear mutations, andthus are detected more sensitively. Mitochondrial mutations can bemonitored over time to detect changes in the amount of exposure topollutants. In addition, the prevalence of the mitochondrial mutation inthe sample indicates whether clonal proliferation has occurred. Finally,the D-loop has been identified as a hotspot of mutations within themitochondrial genome.

Mitochondrial mutations are determined with reference to wild-type humanmitochondrial sequence. Sequence information can be found at the websitehttp://www.gen.emory.edu/mitomap.html and at SEQ ID NO: 1. However, somedifferences between a sample sequence and a documented wild-typesequence can be polymorphisms, not mutations. Table 3 provides a numberof new polymorphisms. Other polymorphisms can be found in references 2and 8. Polymorphisms can be distinguished from somatic mutations bycomparing the sequence in the sample to the corresponding sequence in anormal body tissue of the same person. If the same variant sequence isfound in the sample as in the normal body tissue it is a polymorphism.Normal tissues can be paraffin-embedded. It has been found by thepresent inventors that mitochondrial DNA which is paraffin-embeddedremains more highly intact and amplifiable than genomic DNA. Amplifiableregions of mitochondrial DNA may be from 10 bp to about 4 kb, desirably2 kb to 4 kb or 10 bp to about 2 kb. Other suitable sources of referencemtDNA are blood, serum, or plasma of the human being tested.

Suitable bodily fluids for testing according to the present inventioninclude saliva, sputum, urine, and bronchoalveolar lavage (BAL). Thesecan be collected as is known in the art. People who are prime candidatesfor testing and supplying such bodily fluids are those who have beenepisodically, periodically or chronically exposed to environmentalpollutants. These include without limitation cigarette smoke, biologicaltoxins, such as aflatoxin, cholera toxin, and botulinum toxin, radiationincluding UV irradiation, industrial wastes, chemicals, water-borne orair-borne pollutants, and drugs. The environmental pollutant can beknown, suspected, or unidentified, as the assay depends on the effectand not on the identity of the pollutant.

The inventors have found that there are certain characteristics of themutations which are found in mitochondrial DNA. Many mutations are foundin sequences which do not encode proteins. These include the D-loopregion (i.e., nucleotides 16024-526), the 16S RNA gene, and the tRNAgenes. Furthermore, even where the mutations do occur in protein codingregions, they often result in silent mutations which do not affect theencoded amino acids. Other regions frequently affected include the genesfor NADH dehydrogenase 4, NADH dehydrogenase 3, NADH dehydrogenase 5,and cytochrome B.

Mutation detection can be done according to any methodology which isknown in the art for determining mutations. These include withoutlimitation, nucleotide sequencing, hybridization, amplification, PCR,oligonucleotide mismatch ligation assays, primer extension assays,heteroduplex analysis, allele-specific amplification, allele-specificprimer extension, SCCP, DGGE, mass spectroscopy, high pressure liquidchromatography, and combinations of these techniques.

Prevalence of a particular mutation according to the present inventioncan be used to monitor clonal expansion. Mutations which are present ingreater than 1% of the mitochondrial DNA present in a sample have mayhave conferred a growth advantage on the cells harboring them. Even ifno growth advantage is conferred by the mutation itself, the mutationserves as a marker for a clone which is expanding relative to thepopulation of cells in the sample. Clonal expansion can be measured overtime to monitor the growth of the clone or to monitor the efficacy ofanti-proliferative agents which can be considered environmentalpollutants, according to the present invention.

The inventors have also found that the presence of subtle mutations inthe mitochondrial genome can be used as a means to trace the presence,spread, metastasis, growth, or recurrence of a tumor in a patient. Suchsubtle mutations include single basepair substitutions, single basepairinsertions, and single basepair deletions. Single basepair substitutionscan be either transitions or transversions, although the former are morefrequent. Detection of such mutations can be useful to screen for theinitial appearance of a tumor as well as the recurrence of a previouslyidentified tumor. The methods are particularly suited to monitoranti-cancer therapy, recurrence, metastasis, and completeness ofsurgical removals.

A single basepair substitution is the substitution of a singlenucleotide base with a different nucleotide base at the same position,with the corresponding substitution of the complementary base on theother strand of the DNA. While any single basepair substitution isconceivable within the scope of the invention, the most frequentlyencountered substitutions are those which are consistent with endogenousoxidative damage, such as T to C or G to A transitions, or which areconsistent with a variety of external carcinogens which cause a varietyof types of mutations. The mutations can appear in protein coding ornon-coding regions or in regions which encode ribosomal or transferRNAs.

The homoplasmic or near homoplasmic property of most mutantmitochondrial genomes from tumors permits the ready detection of suchmutations within a sample of mitochondrial DNA from a patient.Homoplasmic mutations are those which appear in essentially all of thecopies of the mitochondrial genome within a given cell or tissue.However, heteroplasmic mutations, which are those appearing in only afraction of the mitochondrial genomes of a cell or tissue, are alsosuitable for use with the invention.

Any cell sample can be tested from a patient who has cancer or issuspected of having cancer. Suitable cell samples include, but are notlimited to, tissue from a growth suspected or known to be cancerous,tissue adjacent to a resection of a tumor, and tissue distant from thesite of a tumor, such as lymph nodes which are suspected of bearingmetastatic cells. Cells can also be obtained from bodily fluids orsecretions, e.g., blood, urine, sputum, saliva, or feces, which maycontain cancerous cells or metastatic cells. Cell samples can also becollected from other bodily secretions and tissues as is known in theart. A cell sample can be collected from suspected or known canceroustissue or from bodily fluids or secretions harboring cancer cells aswell as from suspected or known normal tissue or bodily fluids orsecretions harboring normal cells.

In order to detect mutations of the mitochondrial genome from a cellsample of a patient, mitochondrial DNA can be isolated from the cellsample using any method known in the art. One way of identifying subtlemutations involves sequencing the mitochondrial DNA. This can be doneaccording to any method known in the art. For example, isolatedmitochondrial DNA can be cleaved using endonucleases into overlappingfragments of appropriate size for sequencing, e.g., about 1-3 kilobasesin length, followed by polymerase chain reaction (PCR) amplification andsequencing of the fragments. Examples of DNA sequencing methods arefound in Brumley, R. L. Jr., and Smith, L. M., 1991, Rapid DNAsequencing by horizontal ultrathin gel electrophoresis, Nucleic AcidsRes. 19:4121-4126 and Luckey, J. A., Drossman, H., Kostihka, T.; andSmith, L. M., 1993, High-speed DNA sequencing by capillary gelelectrophoresis, Methods Enzymol. 218:154-172. Amplification methodssuch as PCR can be applied to samples as small as a single cell andstill yield sufficient DNA for complete sequence analysis. The combineduse of PCR and sequencing of mitochondrial DNA is described in Hopgood,R., Sullivan, K. M., and Gill, P., 1992, Strategies for automatedsequencing of human mitochondrial DNA directly from PCR products,Biotechniques 13:82-92 and Tanaka, M., Hayakawa, M., and Ozawa, T.,1996, Automated sequencing of mitochondrial DNA, Methods Enzymol.264:407-21.

Mutations can first be identified by comparison to sequences present inpublic databases for human mitochondrial DNA, e.g., athttp://www.gen.emory.edu/mitomap.html and at SEQ ID NO: 1. Any singlebasepair substitution identified in the sample DNA compared to a normalsequence from a database can be confirmed as being a somatic mutation asopposed to a polymorphic variant by comparing the sample mitochondrialDNA or sequences obtained from it to control cell mitochondrial DNA fromthe same individual or sequences obtained from it. Control cells areisolated from other apparently normal tissues, i.e., tissues which arephenotypically normal and devoid of any visible, histological, orimmunological characteristics of cancer tissue. A difference between thesample and the control identifies a somatic mutation which is associatedwith the tumor.

An alternative to serially sequencing the entire mitochondrial genome inorder to identify a single basepair substitution is to use hybridizationof the mitochondrial DNA to an array of oligonucleotides. Hybridizationtechniques are available in the art which can rapidly identify mutationsby comparing the hybridization of the sample to matched and mismatchedsequences which are based on the human mitochondrial genome. Such anarray can be as simple as two oligonucleotide probes, one of whosesequence matches the wild-type or mutant region containing the singlebase substitution (matched probe) and another whose sequence includes asingle mismatched base (mismatch control probe). If the sample DNAhybridizes to the matched probe but not the mismatched probe, it isidentified as having the same sequence as the matched probe. Largerarrays containing thousands of such matched/mismatched pairs of probeson a glass slide or microchip (“microarrays” or “gene chips”) areavailable which are capable of sequencing the entire mitochondrialgenome very quickly. Such arrays are commercially available. Reviewarticles describing the use of microarrays in genome and DNA sequenceanalysis and links to their commercial suppliers are available atwww.gene-chips.com.

The invention can be used to screen patients suspected of having cancerfor the presence of tumor cells. A cell sample is first obtained from asuspected tumor of the patient, or is obtained from another source suchas blood or lymph tissue, for example, if metastasis is suspected. Thecell sample is tested to determine the presence of a single basepairmutation in mitochondrial DNA from the cell sample using the techniquesoutlined above. Optionally, a cell sample from normal, non-cancerouscells or tissue of the patient is also obtained and is tested for thepresence or absence of a single basepair mutation in mitochondrial DNA.If a single basepair mutation is determined which is not present in acell sample from normal tissue of the patient, then the mutation is asomatic mutation and the presence of tumor cells in the patient isindicated. If one or more single basepair mutations are determined inthe mitochondrial genome of the cell sample of the patient, then thepatient is identified as having a tumor. As in any diagnostic techniquefor cancer, to confirm or extend the diagnosis, further diagnostictechniques may be warranted. For example, conventional histologicalexamination of a biopsy specimen can be performed to detect the presenceof tumor cells, or analysis of a tumor-specific antigen in a blood ortissue sample can be performed.

The method outlined above can be practiced either in the situation wherethe somatic mutation is previously known or previously unknown. Themethod can be practiced even in the absence of prior knowledge about anyparticular somatic mutation. The method can also be carried outsubsequent to the discovery of a somatic mutation in a mitochondrialgenome of a cell of the patient or of another patient. In this case, aprevious association of the somatic mutation with the presence of atumor in the patient or in another patient strongly indicates thepresence of tumor cells in the patient. It may also indicate therecurrence of a tumor or the incomplete prior removal of canceroustissue from the patient.

The effectiveness of therapy can be evaluated when a tumor has alreadybeen identified and found to contain a single basepair substitution inthe mitochondrial genome. Once a single basepair mutation has beenidentified in the mitochondrial DNA of a tumor of the patient, furthertumor cells can be detected in tissue surrounding a resection or atother sites, if metastasis has occurred. Using the methods outlinedabove, the recurrence of the tumor or its incomplete removal can beassessed. Similarly, if a tumor has been treated using a non-surgicalmethod such as chemotherapy or radiation, then the success of thetherapy can be evaluated at later times by repeating the analysis. Thestep for determining the presence of a single basepair mutation in amitochondrial genome of a cell sample of a patient can be performed 1,2, 3, 4, 5, 6, 8, 10, or more times in order to monitor the developmentor regression of a tumor or to monitor the progress or lack of progressof therapy undertaken to eliminate the tumor.

Upon repeated analyses, the step for determining the presence of asingle basepair mutation is simplified, because only a well defined andlimited region of the genome need be sequenced. Using the hybridizationmethod, for example, it is possible to evaluate the presence of themutation with only a single matched/mismatched pair of oligonucleotideprobes in the array. In the event that a mixture of genotypes isobserved, it is possible to obtain quantitative information about therelative amount of each mitochondrial genotype using techniques known tothe art, e.g., hybridization. Quantitative analysis can reveal changesin the relative proportion of tumor to normal cells in a tissue overtime or in response to therapy.

The following examples are provided to demonstrate certain aspects ofthe invention but they do not define the scope of the invention.

EXAMPLE 1

This example demonstrates detection of mt mutations in tissue samples.

To determine whether mt mutations could be identified in cancer otherthan colorectal cancer, we studied primary bladder (n=14), head and neck(n=13), and lung (n=14) tumors (5). Eighty percent of the mt genome ofall the primary tumor samples was PCR-amplified (6) and sequencedmanually (FIG. 1). Tumor mtDNA was compared to mtDNA from paired bloodsamples in all cases, and mtDNA from corresponding normal tissue whenavailable (7). Of the 292 sequence variants detected, 196 werepreviously recorded polymorphisms (2, 8), while 57 were novelpolymorphisms (Table 3). The remaining 39 variants were acquired(somatic) mutations identified in 64% (9/14) of the bladder cancer, 46%(6/13) of the head and neck cancer, and 43% (6/14) of the lung cancerpatients (Table 2). Most of these mutations were T-to-C and G-to-A basetransitions, indicating possible exposure to ROS-derived mutagens (9).Similar to the previous observation by Polyak et al. (Table 1; 4), themajority of the somatic mutations identified here were also homoplasmicin nature. In addition, several of the bladder and head and neck cancersstudied here (Table 2) had multiple mutations implying possibleaccumulation of mtDNA damage.

In the bladder tumors, mutation hot spots were primarily in the NADHdehydrogenase subunit 4 (ND4) gene (35%), and in the displacement-loop(D-loop) region (30%). The D-loop region is a critical site for bothreplication and expression of the mt genome since it contains theleading-strand origin of replication and the major promoters fortranscription (10). Many (73%) of the mutations identified withinprotein-coding regions were silent, except for a (Val→Ala) substitutionin the NADH dehydrogenase subunit 3 (ND3) and a 7-amino-acid deletion incytochrome b (Cyt b). The D-loop region was also commonly mutated inhead and neck cancer (67%). Two of the head and neck tumors (22%)contained mutations in the ND4 gene at nucleotide pairs (nps) 10822 and11150, resulting in amino acid substitutions of Thr→Met and Ala→Thr,respectively. A similar tendency was observed in lung cancers,demonstrating a high concentration of mutations in the D-loop region(70%).

EXAMPLE 2

This example demonstrates detection of mt mutations in bodily fluids.

We hypothesized that the homoplasmic nature of these mutations wouldmake them readily detectable in paired bodily fluids. To test this, weextracted and directly amplified mtDNA from urine samples from patientsdiagnosed with bladder cancer. All three corresponding urine samplesavailable in this study contained the mutant mtDNA derived from tumortissues. For example, the mtDNA from a urine sample of bladder cancerpatient #799 showed the same nucleotide transition (G→A) as seen in thetumor (FIG. 2A). In all cases, the urine sample contained a relativelypure population of tumor-derived mtDNA, comparable to that of themicro-dissected tumor sample. Consistent with this observation, salivasamples obtained from head and neck cancer patients contained nodetectable wild-type (WT) signals (FIGS. 2B, and 2C). By sequenceanalysis alone, we were able to detect mtDNA mutations in 67% (6/9) ofsaliva samples from head and neck cancer patients. In lung cancer cases,we were initially unable to identify mutant bands from pairedbronchoalveolar lavage (BAL) fluids because of the significant dilutionof neoplastic cells in BAL fluid (11), (FIG. 2D). We, thus, applied amore sensitive oligonucleotide-mismatch ligation assay to detect mutatedmtDNA. As shown in FIGS. 3A and 3B, both lung cancer mutations (arrows)were confirmed in tumor mtDNA with more dilute signals in thecorresponding BAL samples, and no signal in the corresponding normaltissues. Again, we detected the majority of mtDNA mutations (8/10) inBAL fluids with the exception of two cases where the ligation assayswere not feasible due to the sequence compositions (16183 and 302 nps)adjacent to the mutations.

EXAMPLE 3

This example demonstrates the enrichment of mitochondrial mutant DNA insamples relative to nuclear mutant DNA.

To quantitate this neoplastic DNA enrichment, we compared the abundanceof mt gene mutations to that of nuclear-encoded p53 mutations in bodilyfluids using a quantitative plaque assay. Nuclear and mt fragments thatcontained a mutated sequence were PCR-amplified and cloned for plaquehybridization (12). Two BAL samples from lung cancer patients werechosen for analysis because they had mutations in both the mt andnuclear genomes. For p53 mutations, the percentages of neoplastic cellsamong normal cells for patients #1113 and #1140 were 0.1 and 3.0%,respectively. Remarkably, the abundance of the corresponding mutatedmtDNA (MT) was 22% and 52% when compared to the wild-type (WT) mtsequence (FIG. 4). This enrichment of mtDNA is presumably due to thehomoplasmic nature of these mutations and the high copy number of mtgenomes in cancer cells. Enrichment was further suggested by ourobservations in head and neck paraffin samples where we were able toPCR-amplify 2-3 kb fragments of mtDNA, whereas we were unable to amplifynuclear p53 gene fragments of over 300 bp.

A role for mitochondria in tumorigenesis was implicated when tumor cellswere found to have an impaired respiratory system and high glycolyticactivity (13,14). Recent findings elucidating the role of mitochondriain apoptosis (15) and the high incidence of mtDNA mutations in coloncancer (4) further support the original hypothesis of mitochondrialparticipation in the initiation and progression of cancer. Althoughfurther investigation is needed to define the functional significance ofmt mutations, our data clearly show that those mutations are frequentand present at high levels in all of the tumor types examined.

The homoplasmic nature of the mutated mitochondria remains puzzling. Itis estimated that each cell contains several hundred-to-thousands ofmitochondria and that each mitochondrion contains 1-10 genomes (16).Conceivably, certain mutated mtDNAs may gain a significant replicativeadvantage. For example, mutations in the D-loop regulatory region mightalter the rate of DNA replication by modifying the binding affinity ofimportant trans-acting factors. Mitochondria that undergo the most rapidreplication are likely to acquire more DNA damage, leading to anaccumulation of mutational events. Although the mechanism may vary forother mutations (such as silent mutations in the ND4 gene), theaccumulation of a particular mtDNA mutation may become more apparentduring neoplastic transformation. Even subtle mtDNA mutations may alsogain significant replicative advantage, perhaps through interactionswith important nuclear factors. Homoplasmic transformation of mtDNA wasobserved in small populations of cells in other non-neoplastic, butdiseased tissues (17), sometimes associated with aging (18). Wehypothesize that, in contrast to classic clonal expansion, the processmay occur as “pseudoclonal” selection where stochastic segregation ofmitochondria (16) together with neoplastic clonal expansion driven bynuclear mutations lead to a homogeneous population of a previously“altered” mitochondrion (FIG. 5).

The large number of mt polymorphisms identified here and elsewhere (2)likely reflects the high mutation rate of mtDNA, which is thought to becaused mainly by high levels of ROS (19). In agreement with this, ourdata imply that constitutive hypervariable areas such as the D-loopregion represent somatic mutational hot spots. As further mutations aretabulated in primary tumors, DNA-chip technology can be harnessed todevelop high-throughput analyses with sufficient sensitivity (20, 21).Due to its high copy number, mtDNA may provide a distinct advantage overother nuclear genome based methods for cancer and environmentalpollutant detection.

LITERATURE CITED

-   1. R. N. Lightowlers, P. F. Chinnery, D. M. Tumbull, N. Howell,    Trends Genet 13, 450 (1997).-   2. MITOMAP: A Human Mitochondrial Genome Database. Center for    Molecular Medicine, Emory University, Atlanta, Ga., USA.    http://www.gen.emory.edu/mitomap.html-   3. D. L. Croteau and V. A. Bohr, J. Biol. Chem. 272, 25409 (1997).-   4. K. Polyak et al., Nature Genet. 20, 291 (1998).-   5. Paired normal and tumor specimens along with blood and bodily    fluids were collected following surgical resections with prior    consent from patients in The Johns Hopkins University Hospital.    Tumor specimens were frozen and micro-dissected on a cryostat so    that the tumor samples contained greater than 70% neoplastic cells.    DNA from tumor sections was digested with 1% SDS/Proteinase K,    extracted by phenol-chloroform, and ethanol precipitated. Control    DNA from peripheral lymphocytes, matched normal tissues, from urine,    saliva, and BAL fluid were processed in the same manner as described    in (11).-   6. Mitochondrial DNAs were amplified using overlapping primers (4)    in PCR buffer containing 6% DMSO. Approximately 5-20 ng of genomic    DNA was subjected to the step-down PCR protocol: 94° C. 30 sec,    64° C. 1 min, 70° C. 3 min, 3 cycles, 94° C. 30 sec, 61° C. 1 min,    70° C. 3 min, 3 cycles, 94° C. 30 sec, 58° C. 1 min, 70° C. 3.5 min    15 cycles, 94° C. 30 sec, 57° C. 1 min, 70° C. 3.5 min, 15 cycles,    and a final extension at 70° C. for 5 min. PCR products were    gel-purified using a Qiagen gel extraction kit (Qiagen) and sequence    reactions were performed with Thermosequenase (Perkin-Elmer) using    the cycle conditions (95° C. 30 sec, 52° C. 1 min, and 70° C. 1 min    for 25 cycles).-   7. Corresponding normal tissues from 4 patients (#874, #915, #1684,    and #1678) were available and DNA was extracted from paraffin    samples as described previously (9).-   8. R. M. Andrew et al., Nature Genet. 23 147, (1999)-   9. J. Cadet, M. Berger, T. Douki, J. L. Ravanat, Rev. Physiol.    Biochem. Pharmacol. 131, 1 (1997).-   8. J. W. Taanman, Biochimica. et. Biophysica. Acta. 1410, 103    (1999).-   9. S. A. Ahrendt et al., J. Natl. Cancer Inst. 91, 332 (1999).-   10. Subcloning of PCR fragments into phage vector was performed    according to the manufacturer's instructions (Stratagene). Titered    plaques were plated and subjected to hybridization using    tetramethylammonium chloride (TMAC) as a solvent. Positive signals    were confirmed by secondary screenings. Oligonucleotides (Oligos)    used for this assay were as follows; for patient #1113, p53 and    mtDNA sequence alterations were detected using oligos containing    either WT-(p53: 5′-GTATTTGGATGTCAGAAACACTT-3′ (SEQ ID NO: 2)/mtDNA:    5′-ACTTCAGGGTCATAAAGCC-3′(SEQ ID NO: 3)) or MT (p53:    5′-GTATTTGGATGTCAGAAACACTT-3′ (SEQ ID NO:    4)/mtDNA:5′-ACTTCAGGGCCATAAAGCC-3′ (SEQ ID NO: 5)) sequences,    respectively. For patient #1140, oligos 5′-ACCCGCGTCCGCGCCATGGCC-3′    (SEQ ID NO: 6) and 5′-ACCCGCGTCCTCGCCATGGCC-3′ (SEQ ID NO: 7) were    used to detect WT and MT sequences, respectively.-   11. O. Warburg, Science 123, 309 (1956).-   12. J. W. Shay and H. Werbin, Mut. Res. 186, 149 (1987).-   13. D. R. Green and J. C. Reed, Science 281, 1309 (1999).-   14. D. C. Wallace, Annu. Rev. Biochem. 61, 1175 (1992).-   15. D. C. Wallace, Proc. Natl. Acad. Sci. USA 91, 8746 (1994).-   16. K. Khrapko et al, N. A. R. 27, 2434 (1999).-   17. C. Richter, J. W. Park, B. N. Ames, Proc. Natl. Acad. Sci. USA    17, 6465 (1988).-   18. M. Chee et al., Science 274, 610 (1996).-   19. S. A. Ahrendt et al., Proc. Natl. Acad. Sci. USA 96, 7382    (1999).-   20. Fragments containing mutations were PCR-amplified and then    ethanol precipitated. For each mutation, discriminating    oligonucleotides that contained the mutated base at the 3′ end were    designed (TAACCATA-3′ (SEQ ID NO: 8) for patient #915 and    TCTCTTACC-3′ (SEQ ID NO: 9) for patient #898). Immediately adjacent    [³²P] end-labeled 3′ sequences (5′-CACACTACTA-3′ (SEQ ID NO: 10) for    patient #915 and 5′-TTTAACCAG-3′ (SEQ ID NO: 11) for patient #898)    were used as substrate together with discriminating oligonucleotides    for the ligation reaction. After a denaturing step of 95° C. for 5′,    the reactions were incubated for 1 hr at 37° in the presence of T4    DNA ligase (Life Technologies), in a buffer containing 50 mM    Tris-Cl, 10 mM MgCl₂, 150 mM NaCl, 1 mM Spermidine, 1 mM ATP, 5 mM    DTT, and analyzed on denatured 12% polyacrylamide gels. [Jen et al.,    Cancer Res. 54, 5523 (1994)].

1. An oligonucleotide probe comprising a sequence of at least 10contiguous nucleotides of a human mitochondrial genome, wherein theoligonucleotide comprises a mutation selected from the group consistingof: a mutation selected from the group consisting of: T→C at nucleotide114; ΔC at nucleotide 302; C→A at nucleotide 386; insert T at nucleotide16189; A→C at nucleotide 16265; A→T at nucleotide 16532; C→T atnucleotide 150; T→C at nucleotide 195; ΔC at nucleotide 302; C→A atnucleotide 16183; C→T at nucleotide 16187; T→C at nucleotide 16519; G→Aat nucleotide 16380; G→A at nucleotide 75; insert C at nucleotide 302;insert C→G at nucleotide 514; T→C at nucleotide 16172; C→T at nucleotide16292; A→G at nucleotide 16300; A→G at nucleotide 10792; C→T atnucleotide 10793; C→T at nucleotide 10822; A→G at nucleotide 10978; A→Gat nucleotide 11065; G→A at nucleotide 11518; C→T at nucleotide 12049;T→C at nucleotide 10966; G→A at nucleotide 11150; G→A at nucleotide2056; T→C at nucleotide 2445; T→C at nucleotide 2664; T→C at nucleotide10071; T→C at nucleotide 10321; T→C at nucleotide 12519; Δ 7 amino acidsat nucleotide 15642; G→A at nucleotide 5521; G→A at nucleotide 12345;G→A at nucleotide 3054; T→C substitution at position 710; T→Csubstitution at position 1738; T→C substitution at position 3308; G→Asubstitution at position 8009; G→A substitution at position 14985; T→Csubstitution at position 15572; G→A substitution at position 9949; T→Csubstitution at position 10563; G→A substitution at position 6264; Ainsertion at position 12418; T→C substitution at position 1967; and T→Asubstitution at position
 2299. 2. An oligonucleotide primer comprising asequence of at least 10 contiguous nucleotides of a human mitochondrialgenome, wherein the oligonucleotide comprises a mutation selected fromthe group consisting of: a mutation selected from the group consistingof: T→C at nucleotide 114; ΔC at nucleotide 302; C→A at nucleotide 386;insert T at nucleotide 16189; A→C at nucleotide 16265; A→T at nucleotide16532; C→T at nucleotide 150; T→C at nucleotide 195; ΔC at nucleotide302; C→A at nucleotide 16183; C→T at nucleotide 16187; T→C at nucleotide16519; G→A at nucleotide 16380; GA at nucleotide 75; insert C atnucleotide 302; insert C→G at nucleotide 514; T→C at nucleotide 16172;C→T at nucleotide 16292; A→G at nucleotide 16300; A→G at nucleotide10792; C→T at nucleotide 10793; C→T at nucleotide 10822; A→G atnucleotide 10978; A→G at nucleotide 11065; G→A at nucleotide 11518; C→Tat nucleotide 12049; T→C at nucleotide 10966; G→A at nucleotide 11150;G→A at nucleotide 2056; T→C at nucleotide 2445; T→C at nucleotide 2664;T→C at nucleotide 10071; T→C at nucleotide 10321; T→C at nucleotide12519; Δ 7 amino acids at nucleotide 15642; G→A at nucleotide 5521; G→Aat nucleotide 12345; G→A at nucleotide 3054; T→C substitution atposition 710; T→C substitution at position 1738; T→C substitution atposition 3308; G→A substitution at position 8009; G→A substitution atposition 14985; T→C substitution at position 15572; G→A substitution atposition 9949; T→C substitution at position 10563; G→A substitution atposition 6264; A insertion at position 12418; T→C substitution atposition 1967; and T→A substitution at position
 2299. 3.-57. (canceled)